Hydrolysis of fats

ABSTRACT

A process for the hydrolysis of liquid fats comprising contacting the fats, in the presence of water at hydrolyzing conditions, with lipase immobilized by adsorption from aqueous solution without pretreatment or pretreatment with a polar solvent on a microporous structure comprising a synthetic hydrophobic thermoplastic polymer selected from the group consisting of aliphatic olefinic polymers, oxidation polymers, ionic polymers and blends thereof. Various embodiments of the invention include immobilized lipase itself and an embodiment that employs a vertical packed column of particles of the immobilized lipase through which the liquid fat and water feed streams may be passed cocurrent or countercurrent, one that employs a horizontally disposed diaphragm that includes a layer of fibers of the immobilized lipase and an embodiment that employs a stirred reactor wherein a suspension of particles of the immobilized lipase is maintained in the reaction medium. The process of the invention has exhibited surprisingly high activity in the hydrolysis of fats and the immobilized lipase possesses significant longevity.

BACKGROUND OF THE INVENTION

The hydrolysis of fats, also known as fat splitting, has long beenaccomplished by the use of high pressure steam. Steam splitter reactionconditions are typically about 250° C. and 750 psig. To maintain theseconditions a boiler is required to supply the high pressure steam aswell as sophisticated pumps capable of pumping feedstock and water intothe steam splitting column at high pressure. The costs involved for thistype of operation, as required for capital investment as well as processcosts such as for energy in the forms of steam, natural gas andelectricity, are of course, very high.

There is thus a significant economic incentive to develop new moreefficient processes for the hydrolysis of fats, and, as has already beendemonstrated in Japan, enzymatic fat splitting is clearly the process ofchoice. The enzyme that catalyzes the hydrolysis of fats is calledlipase, or more formally E.C. 3.1.1.3 glycerol-ester hydrolase. Theoverall chemistry of the reaction is shown below for a typicaltriglyceride: ##STR1##

It may be noted that the above reaction actually proceeds via stepwisehydrolysis of the acyl groups on the glyceride, so that any given time,the reaction mixture contains not only triglyceride, water, glycerol,and fatty acid, but also diglycerides and monoglycerides. Furthermorethe reaction is reversible. The reverse reaction between an alcohol anda fatty acid to form an ester is called esterification. To force theforward reaction to completion, it is necessary to remove one of theproducts from the reaction mixture. This task is made easy by the factthat the reaction actually takes place in a biphasic medium. Thetriglyceride and fatty acid form an oil layer, while the water andglycerol form an aqueous layer. Thus the hydrolysis is easily forced tocompletion in the splitter by removing the glycerol in the sweetwater.

Lipase can be isolated from several sources soil, plants, animals, ormicroorganisms. However, there are important differences in thesubstrate specificity of the lipases harvested from different sources.For example, porcine pancreatic lipase is position specific for theterminal (sn-1 and sn-3) ester bonds on the triglyceride. Lipase fromseveral microbes, such as Rhizopus arrhizus and Mucor miehei also showthe same positional preference for the end acyl groups. Another type ofspecificity is exhibited by the lipase secreted by Geotrichum candida.This lipase preferentially liberates unsaturated fatty acid groupscontaining a cis double bond in the 9-position of the acyl group, suchas oleic and linoleic acids.

A third type of substrate specificity shown by some lipases is that ofchain length. Pregastric esterases of lamb, goat, and kid selectivelysplit shorter carbon chain length acyl groups (C₄ -C₈). These lipases,are used in the manufacture of Italian cheeses. The distinctive flavorof these cheeses is caused by the release of short chain fatty acids.Lipase from Aspergillus niger has also shown a similar specificity forshorter chain lengths.

Lipases that show no substrate specificity and are thus random in theirattack on the glyceride molecule also exist. This is the type of enzymecatalyst that is needed for the fat splitting reaction. The mostprevalent nonspecific lipase is isolated from the yeast Candidacylindracea, which has been reclassified recently to Candida rugosa.Pseudomonas type bacteria have also been found to excrete a nonspecificlipase.

There are many references concerned with enzymatic fat hydrolysis. InHousehold & Personal Products Industry, August 1981, Page 31, there wasdisclosed the use of lipase in an enzymatic process in which oil and fatare separated into fatty acid and glycerine. Likewise, the followingreferences discuss various methods of effecting enzymatic fathydrolysis:

A. Mitsutani, Research & Development Review Report No. 27, Applicationof Microbiological Technology to Chemical Process, Nippon ChemtecConsulting, Inc., March 1984.

W. Linfield, R. Barauskas, L. Sivieri, S. Serota, R. Stevenson,"Enzymatic Fat Hydrolysis and Synthesis", JAOCS, 61 (2), Feb. 1984, pp.191-195.

W. Linfield, D. O'Brien, S. Serota, R. Barauskas, "Lipid-lipaseInteractions I. Fat Splitting with Lipase from Candida rugosa", JAOCS,61 (6), June 1984, pp. 1067-1071.

G. Benzonana, S. Esposito, "On the Positional and Chain Specificities ofCandida Cylindracea Lipase", Biochim, Biophys. Acta, 231 (1971) pp.15-22.

None of the above references discusses the use of immobilized enzymes.The immobilization of enzymes on solid supports has advantages that havelong been recognized. A particular advantage is that the immobilizedenzyme remains bonded to the support rather than passing through withthe substrate upon which it is acting so that there is no need torecover the enzyme from the substrate and so that the enzyme remains inthe support where it may be reused.

Japanese Patent Publication JP No. 84091883 (Abstract No. 84-168208) ofMay 5, 1984 discloses that an immobilized enzyme may be produced bybringing an aqueous solution of enzyme into contact with a poroussynthetic hydrophobic adsorbent. Examples given of adsorbent materialsare styrene and methacrylic acid ester. The reference, however, gives nohint to the hydrolysis of fats, nor to lipase as the enzyme.

Russian Patent Publication No. SU 804647 (Abstract No. 83249D) of Feb.15, 1981 discloses crosslinked porous styrene polymers used as activityenhancing carriers for immobilized enzymes, but also does not hint tothe composition, process, methods or apparatus of the present invention.

There is also art that teaches the hydrolysis of fats by use ofimmobilized lipase. In Chemical Week, Vol. 133, No. 22, Nov. 30, 1983,on page 33, it is generally mentioned that a number of useful enzymesmay be immobilized by locking them to a carrier by adsorption,crosslinking or covalent bonding, and on page 34 there is mention thatlipase may be used to hydrolyze fat, but there is no teaching in thisreference of polymeric carriers, and there is a warning on page 33 thatan enzyme free in solution and the same enzyme locked to a carrier donot behave the same.

In J. Lavayre, J. Baratti, "Preparation and Properties of ImmobilizedLipases", Biotech & Bioengr., 24 (1982), pp. 1007-1013, hereinafterreferred to as "Lavayre et al", there is discussed the use of lipaseimmobilized by adsorption onto a hydrophobic support for the hydrolysisof olive oil. The Lavayre et al article, however, states that whenpurified pancreatic lipase was used, the specific activity of theimmobilized enzyme was 17 to 25% that of the soluble enzyme.Furthermore, the only support used in the hydrolysis tests was theiodopropyl derivative of porous glass (Spherosil).

The use of lipase immobilized onto polyacrylamide beads for thehydrolysis of triglyceride is discussed in "Bell, Todd, Blain, Patersonand Shaw", Hydrolysis of Triglyceride by Solid Phase Lipolytic Enzymesof Rhizopus arrhizus in "Continuous Reactor Systems", Biotech &Bioengr., 23 (1981), pp. 1703-1719, and in Lieberman and Ollis,"Hydrolysis of Particulate Tributyrin in a Fluidized Lipase Reactor",Biotech & Bioengr., 17 (1975), pp. 1401-1419. In those references,however, the immobilization is effected by covalent bonding (e.g.diazonium intermediate), not adsorption. The results were a significantdecrease in the activity of the immobilized as compared to the freeenzyme.

The hydrolysis of fats with lipase is a reversible reaction and thereare teachings in the art of methods of producing fats by reacting afatty acid with water and glycerol in the presence of lipase. One suchreference is M. M. Hoq, T. Yamane, S. Shimizu, T. Funada, S. Ishida,"Continuous Synthesis of Glycerides by Lipase in Microporous MembraneBioreactor", JAOCS, 61 (4), April 1984, pp 776-781, hereinafter referredto as "Hoq et al". Hoq et al advises against the use of immobilizedlipase for the stated reason that its activity is commonly only severalpercent of the original activity of the free lipase. Hoq employs adevice, it refers to as a bioreactor, which comprises supportedhydrophobic microporous membrane, in particular one made frompolypropylene, that is placed at the interface of an upper phase offatty acid and lower phase of a solution of glycerol, water and lipase.The reactants and lipase come into contact at the interface of the twophases thereby causing the reaction, the glycerides diffusing back intothe bulk flow of the fatty acid phase.

The present invention is based on the surprising discovery that lipaseimmobilized on certain porous polymeric supports in a certain mannerloses very little of its fat hydrolysis activity as compared to solublelipase, notwithstanding teachings of prior art such as Lavayre et al andHoq et al that immobilization of lipase causes such activity to diminishto a small fraction of the free lipase.

SUMMARY OF THE INVENTION

It is a primary objective of the present invention to obtain acomposition comprising an immobilized lipase that has a uniquesuitability for use in a process for the hydrolysis of fats. Anotherobjective is to obtain a process for the hydrolysis of fats using suchimmobilized lipase without significant sacrifice of lipase activity ascompared to free soluble lipase. Other objectives are to provide amethod for the immobilization of the lipase as well as means and deviceswhich use the immobilized lipase of the invention in a practical andefficient manner.

Accordingly, the present invention, in a first embodiment is acomposition comprising lipase immobilized by adsorption from aqueoussolution on a microporous structure comprising a synthetic hydrophobicthermoplastic polymer selected from the group consisting of aliphaticolefinic polymers, oxidation polymers, ionic polymers and blendsthereof. The structure is not pretreated prior to the adsorption or ispretreated by wetting with a polar water miscible organic solvent inwhich the polymer is insoluble and which does not deactivate the lipase.

In a second embodiment, the present invention comprises a method for theimmobilization of lipase on a microporous structure comprising asynthetic hydrophobic thermoplastic polymer selected from the groupconsisting of aliphatic olefinic polymers, oxidation polymers, ionicpolymers and blends thereof. The immobilization is effected eitherwithout pretreatment of the structure or is pretreated by first wettingthe polymer with a polar water miscible organic solvent in which suchpolymer is insoluble and which does not deactivate the lipase. Thesupport is then soaked in a dilute aqueous solution of the lipase.

In a third embodiment, the present invention comprises a process for thehydrolysis of liquid fats. The process comprises contacting liquid fats,in the presence of water at hydrolyzing conditions, with lipase,immobilized by adsorption from aqueous solution on a microporousstructure. The structure comprises a synthetic hydrophobic thermoplasticpolymer selected from the group consisting of aliphatic olefinicpolymers, oxidation polymers, ionic polymers and blends thereof. Thestructure may be pretreated prior to adsorption only by wetting with apolar water miscible organic solvent in which the polymer is insolubleand which does not deactivate the lipase. Pretreatment, however, is notessential.

In a fourth embodiment, the present invention comprises a process forthe hydrolysis of liquid fats comprising contacting the fats in thepresence of water at hydrolyzing conditions with lipase immobilized byadsorption from aqueous solution on a microporous structure comprising asynthetic hydrophobic thermoplastic polymer selected from the groupconsisting of aliphatic olefinic polymers, oxidation polymers, ionicpolymers and blends thereof. The structure is either not pretreated oris pretreated prior to adsorption only by wetting with a polar watermiscible organic solvent in which the polymer is insoluble and whichdoes not deactivate the lipase. The contacting is effected by means of acolumn packed with a powder of the structure on which the lipase isimmobilized. The powder in the cocurrent embodiment is preferably fromabout 150 to about 450 micron particle size.

In a fifth embodiment, the present invention is a process for thehydrolysis of fats. The process comprises maintaining a lower liquidphase of fats and an upper liquid phase which comprises water. Thephases are separated at their interface with a horizontally disposeddiaphragm which comprises three layers. The bottom most layer is ahydrophobic filter cloth. The middle layer is fibers of a supportcomprising a synthetic microporous thermoplastic polymer having lipaseimmobilized thereon. The top most layer of the diaphragm is a retainingmeans capable of maintaining the fibers of the middle layer in place.The fats flow upward through the bottom layer and into contact with thesupported lipase of the middle layer, where in the presence of waterfrom the upper phase, and, at hydrolyzing conditions, the hydrolysis ofthe fats occurs. The fatty acids product of the hydrolysis rises to thesurface of the upper phase to form a separate uppermost phase. Theglycerol product of the hydrolysis dissolves in the upper phase and thefatty acids are removed as the uppermost phase and glycerol products arerecovered from the upper phase. Additional fats and water are added asrequired to maintain the desired inventory of each.

In a sixth embodiment, the present invention is a process for thehydrolysis of fats which comprises maintaining a suspension comprisinglipase immobilized by adsorption from aqueous solution on particles of amicroporous structure. The process employs a synthetic hydrophobicthermoplastic polymer which is selected from the group consisting ofaliphatic olefinic polymers, oxidation polymers, ionic polymers andblends thereof, in a liquid reaction mixture. The structure may bepretreated prior to the adsorption by wetting with a polar watermiscible organic solvent in which the polymer is insoluble and whichdoes not deactivate the lipase. Pretreatment, however, is not essential.The reaction mixture comprises fats and water and is maintained by thecontinuous addition thereto of a stream of liquid fats and water. Aportion of the reaction mixture containing reaction products whichcomprise fatty acids and a glycerol solution is continuously withdrawn.

A seventh embodiment of the present invention is an apparatus related tothe above fifth embodiment comprising a diaphragm suitable for use inthe hydrolysis of fats comprising:

a. a first layer consisting of a hydrophobic filter cloth havingopenings from about 3 to about 5 microns in size;

b. a second layer adjacent to said first layer comprising fibers of ahydrophobic microporous thermoplastic polymer selected from the groupconsisting of aliphatic olefinic polymers, oxidation polymers, ionicpolymers and blends thereof, having lipase immobilized on the fibers byadsorption from an aqueous solution either without pretreatment orfollowing pretreatment of the fibers only by wetting with a polar watermiscible organic solvent in which the polymer is insoluble and whichdoes not deactivate the lipase; and

c. a third layer adjacent to the side of said second layer opposite saidfirst layer comprising a retaining means capable of maintaining thefibers of the second layer in place.

Other embodiments of the present invention encompass details aboutprocess flow schemes, reaction conditions and materials compositions,and mechanical details all of which are hereinafter disclosed in thefollowing discussion of each of the facets of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are, respectively, elevation views of the embodiment ofthe invention comprising cocurrent and countercurrent flow packedcolumns.

FIG. 3 is illustrative of the embodiment of the invention employing amulti-layered diaphragm.

FIG. 4 is illustrative of a multi-diaphragm embodiment of the invention.

FIG. 5 is illustrative of the stirred reaction embodiment of theinvention.

FIGS. 6 through 11 comprise graphic presentations of data obtained asdescribed in the examples.

DESCRIPTION OF THE INVENTION

There has long been an interest and unfulfilled need to immobilizelipase for use in fat splitting, particularly Candida lipases in view oftheir high activity for that purpose. A primary reason for such need isthe high cost of such lipase which dictates that it be used in animmobilized form so that it can be reused many times. Otherwise, theenzymatic fat splitting process, at least in view of current costs ofenzymes and energy, could never be competitive with non-catalytic highpressure steam fat splitting.

As mentioned above, however, the prior art teaches that theimmobilization of lipase causes its activity to diminish to a smallfraction of free lipase. The present inventors, therefore, were greatlysurprised to find that little if any of such activity was lost when thelipase was immobilized on an appropriate porous polymeric support.

At about the time of the discovery of the surprising utility of lipaseimmobilized on porous polymeric support for fat splitting was thediscovery that when immobilization was carried out in a certain way, aneven superior product was obtained. The method then thought to be bestfor immobilization involved pretreating the polymer with a metal saltsolution (e.g. stannous chloride) and/or a long chain cationic solution(e.g. a salt of N-coco-1,3-diaminopropane or trimethyltallowammoniumchloride). The enzyme was then immobilized onto the petreated polymer.When immobilization experiments started with lipase and polymericsupports for the splitting of fats, however, it was noted that not onlydid the pretreatment have no positive effect on the activity of theimmobilized lipase, a lack of pretreatment (other than wetting thesupport with a polar solvent) was found to be beneficial.

The method of immobilization found to be most effective is to simplysoak the support in a dilute aqueous solution of the lipase, or,optionally, first wet the polymer structure with a polar water miscibleorganic solvent in which the polymer is insoluble and which does notdeactivate the lipase. The concentration of the lipase solution mayvary. A dilute solution is about 35 lipase units per ml., whileconcentrated would be about 500 units per ml. A unit is defined as theamount of lipase required to produce one micro-mole of fatty acid perminute from an olive oil substrate under the conditions of the assay,typically at a pH of 7° and 35° C. The pH of the lipase solution is notimportant, and may be buffered to any value, with an optimum betweenabout 4 to about 7.

The term "polar" as used herein shall mean the property of having adipole moment of at least 0.1 debye. The term "water miscible" shallmean capable of mixing in any ratio with water without separation ofphases. The term "insoluble" shall mean a solubility in the solvent inquestion of not greater than 0.1 g/l. The term "deactivate" shall meanthe loss of the ability to catalyze the hydrolysis reaction.

The hydrophobic microporous cellular polymer selected must be amicroporous (about 0.1-500 micron average pore diameter) synthetichydrophobic thermoplastic polymer selected from the group consisting ofaliphatic olefinic polymers, oxidation polymers, ionic polymers andblends thereof. Polypropylene and polyethylene are examples of nonionicpolymers. The binding of lipase to the nonionic polymers is byhydrophobic adsorption. A minimum hydrophobicity is essential for thenonionic polymers. Nonionic polymers effective for the presentinvention, and having a sufficient degree of hydrophobicity, areconsidered to be those having a surface tension less than 41 dynes/cmwhich includes polyethylene and polypropylene. For the ionic polymers,e.g. Surlyn®, the binding of lipase to polymer may no longer be simplyhydrophobic bonding, but rather complicated by ionic interactions. Thus,surface tension would no longer be a relevant parameter. For thesepolymers for which surface tension is not a relevant parameter, the term"hydrophobic" may have its commonly understood meaning as defined inHackh's Chemical Dictionary, 4th Edition, i.e. a substance that does notadsorb or absorb water.

The ideal microporous structure for the polymeric supports and method ofobtaining such structure are as disclosed in U.S. Pat. Nos. 4,247,498and 4,519,909 issued to Castro, both incorporated by reference herein intheir entirety. Those patents disclose micorporous cellular polymerstructures known by the trademark Accurel® which are marketed by EnkaAmerica Incorporated, 1827 Walden Office Sq., Suite 480, Schaumburg,Ill. 60195. Accurel® structures may be characterized in one of threeways:

1. a cellular microporous structure which comprises a plurality ofsubstantially spherical cells having an average pore diameter from about0.5 to about 100 microns, distributed substantially uniformly throughoutthe structure, adjacent cells being interconnected by pores smaller indiameter than the microcells, the ratio of the average cell diameter tothe average pore diameter being from about 2:1 to about 200:1, the poresand the cells being void.

2. A cellular microporous structure which is cellular and ischaracterized by a C/P ratio of from about 2 to about 200, an S value offrom about 1 to about 30, and an average cell size from about 0.5 toabout 100 microns.

3. An isotropic microporous structure that is characterized by anaverage pore diameter of from about 0.1 to about 5 microns and an Svalue of from about 1 to about 10.

In numbers 2 and 3 above "C" means average diameter of cells, "P" theaverage diameter of the pores, and "S" is the sharpness factor,determined by use of a Micromeritics Mercury Penetration Porosimeter,and defined as the ratio of the pressure at which 85 percent of themercury penetrates the structure to the pressure at which 15 percent ofthe mercury penetrates.

MEANS TO ACHIEVE THE CONTACTING OF THE REACTANTS WITH THE IMMOBILIZEDLIPASE

One means to achieve the contacting of the liquid fats with theimmobilized lipase is a column packed with discrete particles of theimmobilized lipase. The fixed bed column reactor is a very common designfor immobilized enzyme reactors. In this type of reactor fats and watermay either be passed cocurrently through the column in a directionparallel to the longitudinal axis of the column, or countercurrentlythrough the column in directions parallel to the longitudinal axis ofthe column. In a packed column, the activity of the lipase may berestored by first flushing the contents of the column with a solventsuitable for the removal of spent lipase and residual fat from theporous polymeric support (e.g. alcohol), then flushing the contents ofthe column with water to remove the solvent, then flushing with a brothof fresh lipase and finally flushing the contents of the column withwater to wash away excess enzyme.

With the cocurrent type flow scheme fats and water are passed into thecolumn at one end and reaction products comprising glycerol and fattyacids are removed at the opposite end. FIG. 1 illustrates the cocurrentfixed bed immobilized lipase reactor including vertical column 1 packedwith bed 2 of particles of immobilized lipase. The particles arepreferably in the form of powder of from about 150 to about 450 micronsin particle size. Liquid fats are passed from tank 3 via conduit 4 andpump 5 into mixing conduit 6, and water is passed from tank 7 viaconduit 8 and pump 9 into mixing conduit 6. In mixing conduit 6 vigorousmixing is effected so as to obtain a fat/water emulsion which passesinto column 1 and one end of bed 2. The reaction mixture is passedthrough bed 2 over a period of time from about 2 to about 10 hours (orlonger, depending on the degree of conversion desired). The reactionmixture exits the top of bed 2 and separates into an aqueous phase 10,also referred to as "sweetwater" since that is where dissolved glyerolproduct accumulates, and fatty acid phase 11. Aqueous phase product mayperiodically or continuously be drawn off via conduit 12 and fatty acidphase product may be drawn off via conduit 13.

FIG. 2 illustrates the countercurrent fixed bed immobilized lipasereactor, including vertical column 20 packed with bed 21 of particles ofimmobilized lipase. Liquid fats are passed from tank 22 via conduit 23,pump 24 and conduit 25 into the bottom of column 20 and first aqueousphase 26. Water is passed from tank 27 via conduit 28, pump 29 andconduit 30 into the top of column 20 and second aqueous phase 31. Thewater and fats will flow countercurrently through bed 21 due to theeffect of gravity and the difference in specific gravity between thephases, the fats being the lighter of the two phases. The temperatureand residence time of the reactants in bed 21 will be about the same asfor the above cocurrent reactor. Fatty acid will accumulate in fattyacid phase 32 and may periodically or continuously be drawn off viaconduit 33, while at the same time sweetwater is drawn from aqueousphase 26 via conduit 34 and pump 35.

Another type of continuous fat hydrolysis reactor is the diaphragmreactor as illustrated in FIG. 3. Fat in a liquid form enters a bottomportion of vessel 40 via conduit 41. Vessel 40 is separated into abottom and top portion by a horizontal diaphragm comprising threelayers. The bottom most layer 42 is a hydrophobic filter cloth. Suitablematerials for filter cloth 42 are PTFE (Teflon) and polypropylene. Themiddle layer 43 comprises fibers of the porous polymeric support onwhich the lipase is immobilized. The fibers that have heretofore beenused averaged about 3-7 microns in diameter. The thickness of the fiberlayer heretofore observed, following compression through use was about2-4 mm. The top most layer 44 is a retaining means capable of keepingthe fibers in place and may comprise any type of screen havingappropriately sized openings. There may be an additional screen 45supporting the entire diaphragm if filter cloth 42 is consideredinadequate for that purpose.

Choice of the hydrophobic filter cloth comprising bottom most layer 42is particularly important. The openings in the cloth must be largeenough to permit an acceptable flow rate of fat through the cloth, butnot so large that water would pour through from above. Filter clothsfound to be effective were Gore-Tex® Expanded PTFE Membranes andLaminates with 3-5 μ openings from W. L. Gore and Associates, Inc. and0.5-1.0 CFM rated (air flow measured at 1/2" H₂ O pressure on a FrazierPermeometer) PP cloth from CrosIble, Inc.

In operation, the diaphragm will serve to separate at their interface alower liquid fat phase 46 and an upper water phase 47. The fats willflow upward through bottom layer 42 and come into contact with thesupported lipase of middle layer 43 where in the presence of the waterfrom the upper phase 47, and with a residence time in the diaphragmitself of from about 20 minutes to about 60 minutes, the hydrolysis ofthe fats will occur. The fatty acids that are formed, rather thanremaining in the fat phase, will advantageously due to their inherentbuoyancy, rise to the surface of the upper phase 47 to form a separateuppermost phase 48. The glycerol product of the hydrolysis will dissolvein the upper aqueous phase 47.

Water may enter the top portion of tank 40 via conduit 49. Fatty acidmay be withdrawn as a product stream via conduit 51 and glycerol viaconduit 50. Stirrer 52 will serve to maintain a reasonably homogeneoussolution in phase 47. Additional fat and water may be added as requiredto maintain the desired inventory of each.

In the process as shown in FIG. 3, the activity of the lipase withrespect to the hydrolysis of the fats may be restored by flushing thediaphragm sequentially with three flushing liquids which enter thediaphragm at top layer 44 and exit through a means provided to bypassthe filter cloth, such as a conduit and valve. The first flushing liquidcomprises a solution of water and a solvent suitable for the removal ofspent lipase from the support. Suitable solvents are the same as thosethat may be used to pretreat the polymer in effecting the initialimmobilization. The second flushing liquid comprises a broth of freshlipase. The third flushing liquid comprises water.

A multi-stage diaphragm reactor is as illustrated in FIG. 4. Each stagein this illustration comprises a separate vessel in which a diaphragm iscontained. Three vessels, 53a, 53b and 53c, are shown, but any number(two or more) may be used. Diaphragms 53a, 53b and 53c, as describedabove, of horizontal orientation are placed in sealed contact with theinterior surface of the walls of vessels 54a, 54b and 54c, respectively.Conduits 55a and 55b connect vessels 53a to 53b and vessels 53b to 53c,respectively, at points immediately above the diaphragms in each vessel.Conduits 55c and 55d connect vessels 53c to 53b and vessels 53b to 53a,respectively, at points above the diaphragms of vessels 53c and 53b andat or near the tops of those vessels to below the diaphragms of vessels53b and 53a. There is a water-glycerol product withdrawal conduit 56connected to vessel 53c immediately above diaphragm 54c. There is afatty acid product withdrawal conduit 57 connected to vessel 53a abovediaphragm 54a and at or near the top of the vessel. There is a waterinlet conduit 58 connected to vessel 53a above diaphragm 54a. There is aliquid fat (oil) inlet conduit 59 connected to vessel 53c belowdiaphragm 54c.

The apparatus of FIG. 4 may be used for that embodiment of the processof the present invention, employing a multiplicity of the diaphragmsdiscussed above with reference to FIG. 3. Since the conversion per passof the diaphragm appears to not be extremely high, multiple stages areconsidered advantageous. In using the multi-diaphragm scheme of theinvention, as shown in FIG. 4, each diaphragm is associated with a stagewhich also includes the lower, upper and uppermost phases as discussedabove. Each stage is contained in a separate vessel. For reason ofsimplicity, various pumps and control valves that one skilled in the artwould understand to be required are not shown in FIG. 4.

In the operation of the apparatus of FIG. 4, the direction of flow ofthe non-aqueous streams from vessels 53c to 53b to 53a via lines 55c and55d is considered "downstream" flow. Conversely, the direction of flowof the aqueous streams from vessels 53a to 53b to 53c via lines 55a and55b is considered "upstream" flow and countercurrent to the non-aqueousstream. The liquid fat to the process is introduced to vessel 53c vialine 59 below diaphragm 54c into the lower phase of the first upstreamstage. The water to the process is introduced to vessel 53a via line 58into the upper phase of the last downstream stage above diaphragm 54a.Although line 58 is shown as passing the water directly into the upperphase in vessel 53a, it may be more convenient to introduce the water atthe top of the vessel into the uppermost phase whereupon it would simplyflow through the uppermost non-aqueous phase, with which it isimmiscible, and thereby, in effect, be introduced into the upper phase.The aqueous stream from the upper phase of each stage in vessels 53a and53b is passed via lines 55a and 55b, respectively, to the upper phasesof vessels 53b and 53c, respectively, while the aqueous stream from theupper phase of the first upstream stage in vessel 53c is withdrawn asthe glycerol product steam via line 56. The non-aqueous stream from theuppermost phases of the stages in vessels 53c and 53b are passed vialines 55c and 55d, respectively, to the lower phases of the stages invessels 53b and 53a, respectively, while the non-aqueous stream from theuppermost phase of the stage in vessel 53a is withdrawn as the fattyacid product stream via line 57.

The embodiment of the present invention illustrated in FIG. 4 is onlyone possible way of achieving a multi-stage configuration. There areother possibilities including a multiplicity of diaphragms in a singlevessel. The FIG. 4 configuration, however, is considered advantageousfrom the standpoint of simplicity of construction of the equipment andits operation.

Yet another type of continuous fat hydrolysis reactor is one asillustrated in FIG. 5 which employs a suspension in the liquid reactionmixture of particles of immobilized lipase. This embodiment of theinvention may be referred to as the "stirred reactor" or "CSTR".

Looking to FIG. 5, fats and water streams are continuously introducedinto vessel 60 via lines 61 and 62, respectively. Particles or a powderof the polymer support on which the lipase is adsorbed are kept insuspension in reaction medium 63 by means of stirrer 64. A portion ofthe reaction mixture containing the fatty acids and glycerol solutionproducts is continuously withdrawn via line 65. The residence time inreaction medium 63 may comprise from about 2 to about 60 hours.

There is preferably a filter 66 associated with line 65 to enablerecovery of the products along with the retention of the particles inthe reaction mixture.

As far as reaction conditions for the fat splitting process of thepresent invention are concerned, the most important consideration isthat the temperature be high enough to enable the fat to be in liquidphase but not so high as to cause deactivation of the lipase.Deactivation of Candida Cylindracea becomes significant at about 50° C.The pH of the reaction mixture is not critical, but it may be maintainedat about 7.0 by use of water reactant buffered with sodium phosphate.

EXAMPLES

The following non-limiting examples will serve to illustrate thepreferred and advantageous method for achieving immobilization of lipaseon a porous polymeric support, the superiority of lipase immobilized onsuch support in a process for the hydrolysis of fats, and variousembodiments of the present invention employing certain devices and flowschemes.

EXAMPLE 1

In this example is it shown that pretreatment of Accurel® prior tolipase immobilization thereon with anything other than a polar solventis disadvantageous. Four samples were prepared of Candida cylindracealipase immobilized on polypropylene Accurel® powder. The Candidacylindracea lipase for this example was obtained from Sigma Chemical Co.as its Type VII Lipase, catalog no. L-1754, with a stated activity of470 U/mg solid. On three of the four samples Accurel® was pretreatedwith various materials. On one sample the immobilization was effected byfirst wetting the polymer with ethanol and then, without furtherpretreatment, soaking the support in a dilute aqueous solution of thelipase. The results obtained are as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Pretreatment        Lipase Activity* (IU/g)                                   ______________________________________                                        1.  Ethanol             103                                                   2.  SnCl.sub.2 + ethanol                                                                              91                                                    3.  N--coco-1,3-diaminopropane +                                                                      83                                                        ethanol                                                                   4.  SnCl.sub.2 + N--coco-1,3-                                                                         76                                                        diaminopropane + ethanol                                                  ______________________________________                                         *Lipase activity units (IU's) are a measure of the micro moles of fatty       acid per minute titrated from an olive oil substrate at a pH of 8 and roo     temperature.                                                             

The results from Table 1 illustrate that not only is pretreatment (otherthan only with a polar solvent) unnecessary, significantly improvedactivity is realized when pretreatment, other than with a polar solvent,is avoided.

EXAMPLE 2

In this example there was an investigation of the use of varioussolvents to prewet Accurel® polypropylene powder (150-450u) before theimmobilization on such powders of lipase (Candida cylindracea) fromsolution. The lipase obtained for this example was from EnzymeDevelopment Corporation, and is known as Enzeco® lipase with a statedactivity of 30,000 U/g. Also investigated was the same powder that wasnot prewetted prior to immobilization. The procedure in each case toobtain the immobilized lipase was as follows:

1. 1 g of powder was prewet with solvent (except where prewetting wasnot to occur);

2. 0.2 g. of the lipase was dissolved in 100 ml. of water buffered to pH7.0 with 0.1M sodium phosphate;

3. the powder was soaked in the lipase solution for thirty minutes; and

4. the resulting immobilized lipase was filtered from the solution andflushed three times with additional quantities of water buffered asabove.

A series of tests was then run wherein each of the immobilized lipasesamples prepared by the above procedure was used to hydrolyze fat. Ineach test 50 ml. of Bleachable Fancy Tallow was stirred in a beaker withthe immobilized lipase and 100 ml of buffered water at 42° C. over a 24hr. period. The results obtained, in terms of % fatty acids in thenon-aqueous product phase, at the end of the 24 hour period, are asgiven in the following Table 2:

                  TABLE 2                                                         ______________________________________                                        Pretreatment    % FA                                                          ______________________________________                                        ethanol         58.0                                                          isopropanol     58.2                                                          methanol        55.9                                                          acetone         51.0                                                          tetrahydrofuran 52.6                                                          none            66.0                                                          ______________________________________                                    

The above results show that high levels of fatty acid may be obtainedeither when pretreatment is effected with a polar solvent, or wherethere has been no pretreatment. In the latter case the highest degree offat hydrolysis is achieved, but as will be discussed in the followingexample, prewetting with a polar solvent greatly facilitates the rate atwhich the lipase may be immobilized.

EXAMPLE 3

To illustrate a further advantage of the method of lipase immobilizationof the present invention, a determination was made of the time requiredto obtain the immobilization of lipase on a polypropylene Accurel® in apowdered form (150-450u) and pretreated with ethanol. Immobilizationcould be effected in about two minutes. Without any pretreatmentimmobilization took about five times as long. Thus, althoughpretreatment with the appropriate polar solvent is not necessary toobtain high activity immobilized lipase, it will enable rapid loading ofthe lipase onto the support which is particularly advantageous with insitu loading and regeneration.

EXAMPLE 4

For this example Candida cylindracea lipase (the same as theaforementioned Enzeco® lipase) was immobilized on untreated Accurel®products (treated only with a polar organic solvent) by simpleadsorption from a buffered enzyme solution. The procedure for a typicalimmobilization was to dissolve 0.20 grams of lipase in 100 millilitersof 0.1M sodium phosphate, pH 7.0 buffer. Next, 1.0 gram of Accurel®powder was prewet with as little 3A ethanol as possible and added to theenzyme solution. After stirring for 5-60 minutes, the immobilizedcatalyst was filtered and then rinsed with several bed volumes ofbuffer. The immobilized lipase was assayed for activity using atriglyceride substrate, such as olive oil or Bleachable Fancy Tallow.

Other materials, besides Accurel® were screened as supports for lipase.Table 3 lists all the enzyme support materials that were screened andlists the fatty acid levels generated during the assay at 6 and 24 hourson a tallow substrate relative to the soluble lipase as a control. Thesurprising result was that the porous polymer powders of the presentinvention performed as vastly superior supports for lipase.Specifically, polypropylene (PP), high density polyethylene (HDPE), andSurlyn® Accurel® powders, the more hydrophobic of the polymers used,were superior supports.

On the other hand, inferior results were obtained from polymers of lowhydrophobicity such as cellulose and nylon (nylon is also a condensationpolymer which is not a genus of polymer included in the invention),olefinic polymers which are not aliphatic (styrene) and non-polymericmaterial as well as non-porous material.

Furthermore, comparison of the immobilized enzyme results with those ofthe soluble lipase control in Table 3 indicates that very little of thelipase activity was lost upon immobilization on these supports. Thisresult is diametrically opposite claims in the literature, as previouslydiscussed, that lipase loses 75-99% of its activity upon immobilization.

                  TABLE 3                                                         ______________________________________                                        Lipase Immobilization Supports                                                                    % FA by                                                                       GPC* Relative                                                                 to Soluble Lipase                                         Support               6 HR      24 HR                                         ______________________________________                                        Johns-Manville                                                                Celite 545              8%       12%                                          Celkate               12                                                      R-600                 15                                                      R-640                 15        19                                            CS-30K                15        17                                            Misc                                                                          Microcystalline Cellulose:                                                    Avicel                16        21                                            Avicel, 28μ        19        23                                            Avicel, 50μ        16        29                                            Ethyl Cellulose       31        52                                            Silica Gel            22        23                                            Kieselguhr            19        30                                            Bentone Clay          12        13                                            Alumina, neutral       8         9                                            CPG-100               42                                                      Glycophase-G          15                                                      Styrene dvb × 4% (non-porous)                                                                 11                                                      Celgard 2500 (Celanese Corporation)                                                                 75        85                                            Amberlite XAD-2 (porous styrene)                                                                     8         9                                            Hercules Profax-PP (non-porous)                                                                     15        14                                            USI Microthene-HDPE (non-porous)                                                                    13        12                                            Tenax (porous oxidation polymer of                                                                  75        82                                            2,6 di-phenylparaphenylene oxide)                                             Versapor 200 (porous acrylic copolymer                                                               9         9                                            cast on nonwoven nylon)                                                       AP-200 (porous acrylic copolymer                                                                     9        11                                            cast on nonwoven polyester)                                                   Accurel Powders                                                               Nylon                 16        18                                            Surlyn                82        88                                            HDPE 150μ          93        98                                            HDPE 150- 450μ     62        76                                            PP - std. grind       73        88                                            PP - Enka             78        88                                            PP - Friable          86        99                                            PP - Friable          73        91                                            PP - Friable          72        88                                            PP - Friable          81        88                                            Control                                                                       Soluble Lipase        100       100                                           ______________________________________                                         *1.0 gm support, 3750 IU's Lipase, 1 hour immobilization; 50 mls BFT, 100     mls buffer, 40° C.                                                

EXAMPLE 5

This example describes the operation of fixed bed column reactors asillustrated in FIGS. 1 and 2.

The fixed bed column reactor is a very common design for immobilizedenzyme reactors. The columns studied for splitting fat contained lipaseimmobilized on some form of Accurel®. Accurel® products used as supportmaterials in the columns were Enka America Incorporated melt blown PPAccurel® fibers, Enka's HDPE Accurel® granules (2-3 mm), and HDPEAccurel® powder (150-450 microns). The lipase was immobilized on theAccurel® by simple adsorption. In all cases, lipase from Candida(Enzeco®, as specified above) was dissolved in 0.1M, pH 7.0, sodiumphosphate buffer, the support was prewet with 3A ethanol, and then thesupport was contacted with the lipase solution. These steps wereperformed either in a batchwise process in a beaker or in the columnreactor itself.

To get an idea of how much of the lipase actually adsorbed on thesupport, samples of the lipase solution before and after contact withthe support and/or samples of the support material before and aftercontact with the lipase were analyzed for lipase content using the ANTEKmodel 707 Chemiluminescent Nitrogen Analyzer. For example, 8.0 g of HDPEAccurel® powder were placed in a glass column, wet with 100 mls of 3Aethanol, rinsed with 200 mls of buffered water, contacted with 100 mlsof lipase solution containing 1.0 g of lipase dissolved in 100 mls ofbuffered water, and then rinsed with 150 mls of buffered water. Analysisof the lipase solution before and after contact with the supportindicated that 64.5% of the lipase was removed from solution. Assumingall the removed lipase was immobilized on the powder, the supportcontained (0.645 g/(8.0+0.645) g)×100%=7.5% lipase w/w.

The fixed bed reactors were glass columns jacketed with plexiglasssleeves. The columns were maintained at 40° C., by circulating waterbaths. The actual column dimensions varied. Continuing the exampleinvolving 8.0 g of HDPE powder, the dimensions of the fixed bed were 1.7cm diameter by 21 cm in length (48 cm³).

Substrate was pumped into the columns (5-20 ml/hr), with a piston pumpto maintain close control over the rate. The substrate most used was25/75 olive oil/ 0.1M, pH 7.0 phosphate buffer. Two column flow patternswere investigated: (1) countercurrent flow of the buffered water (enterscolumn at top) and olive oil (enters at bottom), and (2) cocurrent flowof the oil and buffered water (both enter at bottom).

FIG. 1 illustrates the cocurrent fixed bed immobilized lipase reactor.Triglycerides (TG) and buffered water were pumped in the bottom of thecolumn. As mentioned earlier, a variety of Accurel® materials were triedas support for the lipase. Table 4 below compares with % FA obtainedfrom three fixed bed cocurrent reactors operated under conditions whichwere similar except for support material:

                  TABLE 4                                                         ______________________________________                                        Support           Weight (gm)                                                                              % FA                                             ______________________________________                                        PP, melt blown fibers                                                                            4         34                                               HDPE granules     10         42                                               HDPE powder        8         94                                               ______________________________________                                    

Since the HDPE powder, 150-450 micron particle size, was clearly asuperior support to a very surprising and unexpected extent as comparedto the fibers, this powder was used in all the subsequent reactorcolumns. Conditions for two of these columns are given below:

    ______________________________________                                                 Column I     Column II                                               ______________________________________                                        Substrate: 25/75 o.oil/buffered                                                                         25/75 o.oil/buffered                                           water          water                                                          Emulsion Feed  Separate Feed                                       Total Flowrate:                                                                          10-15 ml/hr    20 ml/hr                                            Temperature:                                                                             40° C.  40° C.                                       Support wt:                                                                              8.0 gm         10.0 gm                                             Bed size:  1.7 cm diameter ×                                                                      2.1 cm diameter ×                                        21 cm length   18 cm length                                        Lipase adsorbed:                                                                         650 mg.        975 mg.                                             ______________________________________                                    

Column I was still in operation after 1100 hours. In this column theolive oil and buffered water were mixed together in a beaker prior toentering the column. Continuous agitation was supplied to the emulsionby a magnetic stirrer. A single pump drew off emulsion to feed thecolumn. The volumes of buffer and oil that flowed out of the column wererecorded daily. Considerable variation was observed in the ratios ofoil:water that were collected from the 25:75 ratio which was supplied tothe emulsion reservoir. Thus, throughout the run of this column, thesubstrate composition was not fixed. The half-life (time for theactivity of the immobilized lipase to decrease by 50%) of theimmobilized lipase in this column was 234 hrs. The results for thiscolumn in terms of % fatty acid in the nonaqueous product over time aregraphically shown in FIG. 6.

Column II was operated for 310 hours before being terminated. Thiscolumn had a higher support load and a higher support:lipase ratio inhopes of increasing the half-life of the lipase. In addition, separatepumps were used to pump the olive oil at 5 ml/hr and the buffered waterat 15 ml/hr in order to maintain a constant substrate composition to thecolumn. Also, in the bottom of the column was a glass frit which wasused to break the oil into smaller droplets before contact with thelipase. This column in spite of the efforts made, achieved a half-lifeof only 157 hrs. The results for this column are graphically shown inFIG. 7.

FIG. 2 illustrates the countercurrent fixed bed immobilized lipasereactor. The column bed contained 10 grams of HDPE Accurel® granules.Lipase was immobilized on the granules prior to packing of the column.The catalyst bed measured 2.5 cm diameter by 26 cm in length.

Buffered water was pumped into the top of the column and out the bottomat 5 ml/hr by using two pump heads on the same pump drive. Olive oil waspumped in the bottom of the column at approximately 10 ml/hr. Problemswere encountered in balancing the flows of the oil and water to maintaina steady state in the column. Yields of only 10-20% FA were obtained onthe olive oil substrate, but it is believed that far better results willbe obtained from this embodiment of the invention once it is optimized.

EXAMPLE 6

A study was made using the fixed bed co-current flow column reactor ofthe effect of maintaining a high glycerol content in the water phase. Itwas observed that such content had a profound effect in maintaining theactivity of the lipase although there is a reduction in the degree offat splitting due to reaction equilibrium effects. FIG. 8 comprises aplot of data of the fat split obtained over an extended period of timewith the reaction medium having various levels of glycerineconcentration, and shows the surprising improvement in lipase longevitythat can be achieved by maintaining a concentration of about 40 wt. %glycerol. Concentrations in excess of 40 wt. % would probably not bepractical because of the low conversion of fats that would be obtained.

EXAMPLE 7

The vertical diaphragm reactor embodiment of the invention is shown inFIG. 3. The reactor was constructed of 6" plexiglass pipe, contained alower olive oil reservoir and an upper buffered water reservoir. Oliveoil was pumped into the lower reservoir and was forced up through thediaphragm that separated the upper and lower reservoirs. The diaphragmwas supported on top and bottom by plastic mesh screens. The diaphragmitself consisted of three layers including the top screen. Olive oilfirst passed through the bottom layer which was a teflon filter clothwhich broke the oil into fine droplets. Next the oil passed up through amiddle layer comprising a pad of lipase immobilized on melt blownpolypropylene Accurel® fibers made by Enka America Incorporated. The fatwas split as it passed through the pad. The fatty acid then rose to thetop of the upper reservoir which was initially filled with 1.8 L of0.05M EDTA buffered water to pH 7.0, to form an uppermost phase.Overhead agitation was supplied to the upper reservoir.

To evaluate the operational stability of the immobilized lipase, thisdiaphragm reactor was run in a batch mode for approximately two months.The reactor contained 5.0 g of melt blown Accurel® polypropylene fibersthat contained approximately 0.3 g. of lipase from Candida (Enzeco® asstated above). Each day, 450 mls of olive oil were fed into the reactorby gravity feed over a 6 hour period. At the end of 6 hours, the fattyacid layer on top of the upper reservoir was analyzed by GPC. The topreservoir was then emptied and filled with fresh buffer the followingday.

The plot of % FA versus time in hours, as shown in FIG. 9, gives anindication of the half-life of the immobilized lipase. Linear regressionof % FA versus time of actual operation indicates a half-life of 223hours.

One can use data from the first day to calculate an estimate of theproductivity of the reactor. On that day 450 mls of 71% FA werecollected to arrive at a production rate of 301 mg FA/hr/cm².

EXAMPLE 8

This example illustrates the stirred reactor embodiment of the presentinvention as shown in FIG. 5.

In a first test run immobilized Candida cylindracea lipase (Maxazyme LPfrom Gist-Brocades N.V.) was prepared in the following manner:

1. 10.0 gm. of HDPE Accurel® powder (150-450 μm diameter) was wettedwith 20.0 ml of 3A ethanol.

2. 1.0 gm of Maxazyme LP lipase was dissolved in 100 ml of 0.1M Na₂ HPO₄in aqueous solution at pH 7 by stirring for 10 min.

3. The lipase solution was centrifuged to remove any unsoluble material.

4. The lipase solution was added to the wetted Accurel® powder andstirred for 30 min.

5. The immobilized Accurel® powder was filtered using a Buchner funneland washed with 300 ml of buffered water before it went into thereactor.

A bench scale reactor was constructed comprising a 500 ml round bottomflask with four necks. Two of the necks were for the inlet of liquidfats (oil) and buffered water, the third was for the product streamoutlet, and the fourth was for an overhead stirrer. A glass wool plugwas placed in the product stream outlet neck.

The 500 ml flask was filled with deionized water. Then the aboveimmobilized enzyme was added. The reactor was assembled, an overheadstirrer started, and two feed pumps were turned on, one for each of thefeed streams with a combined flow rate of 12 ml/hr. and a volume ratioof oil to water of 4.5/7.5. The reaction was at room temperature.

The results of the first test run are shown in FIG. 10 in terms of fattyacid concentration in the non-aqueous product phase vs. time. The figureshows a rapid initial fall-off in conversion of fats to a rather lowconstant level.

The first test run was repeated except that 2.0 g. of lipase immobilizedon 20.0 g. of support were added to the reactor. All other conditionsand details of the procedure and apparatus remained the same. Theresults are shown in FIG. 11.

The results of FIG. 11 are startling. Unlike in the first test run whereless amount of lipase was present, in the second run there was noinitial fall-off of conversion and almost no fall-off of conversion evenafter 600 hours of operation were approached. There thus appears to bean amazing and unexpected criticality in a minimum amount of lipase thatmust be present in the reactor mixture for optimum effect.

Since it was determined that only about 65% of lipase initiallyintroduced into the CSTR reactor is retained on the support within ashort time after initiation of the test runs, the critical lipaseconcentration is calculated to be about 2.5 g per liter of reactorvolume for the oil charge rate of 4.5 ml/hr, or 556 grams per liter ofreactor volume per liters per hour of liquid fat charge.

EXAMPLE 9

This example provides a further comparison of the performance of thepreviously discussed embodiments of the present invention comprising thediaphragm, cocurrent fixed bed and stirred tank reactors. In each casethe embodiment used was the one known to be the optimum, including thediaphragm as described in Example 7, the cocurrent fixed bed as inExample 5 with the HDPE Accurel® Powder and the stirred tank andimmobilized lipase on Accurel® as described in Example 8

The results of the comparison are shown in Table 5 where the half-life(in hours) and productivity (pounds fatty acid produced per pound ofimmobilized lipase) are given for each embodiment. Two runs were madefor each of the fixed bed and stirred reactor embodiments.

The results show that the diaphragm clearly comprises the best mode ofthe process of the invention at least as far as productivity isconcerned. In the course of the half-life of the immobilized lipase inthe diaphragm, the productivity of the diaphragm was over five timesthat of the best run of the stirred reactor and over ten times that ofthe fixed bed reactor.

It was also observed that the production rate of the diaphragm in termsof mg/hr. per cm² of fatty acid produced was, depending on the choice ofhydrophobic filter cloth, as high as 301.

                  TABLE 5                                                         ______________________________________                                        IMMOBILIZED LIPASE REACTOR SUMMARY                                            Olive Oil Substrate                                                            Type       Half-Life (hours)                                                                           ##STR2##                                            ______________________________________                                        Diaphragm  223           1695                                                 Fixed Bed  378           119                                                  (Powder columns)                                                                         207            88                                                  Stirred Reactor                                                                          348           342                                                             468           274                                                  ______________________________________                                    

As can be seen from the above examples, and corresponding FIGS. 6through 11, regardless of the embodiment of the invention employed,whether it be a packed column reactor with cocurrent or countercurrentflow, a diaphragm reactor or a stirred reactor, not only is the initialactivity of the immobilized lipase comparable to that of free lipase,the activity remains usefully high for a significant time. Enzymatic fatsplitting has thus become an economic reality.

What is claimed is:
 1. A composition comprising lipase immobilized byadsorption from aqueous solution on a microporous structure comprising asynthetic hydrophobic thermoplastic aliphatic olefin polymer selectedfrom the group consisting of aliphatic olefinic polymers, oxidationpolymers, ionic polymers and blends thereof, said structure not beingpretreated prior to said adsorption or being pretreated only by wettingwith a polar water miscible organic solvent in which said polymer isinsoluble and which does not deactivate said lipase.
 2. The compositionof claim 1 wherein said microporous structure is cellular and comprisesa plurality of substantially spherical cells having an average diameterfrom about 0.5 to about 100 microns, distributed substantially uniformlythroughout the structure, adjacent cells being interconnected by poressmaller in diameter than said microcells, the ratio of the average celldiameter to the average pore diameter being from about 2:1 to about200:1, said pores and said cells being void.
 3. The composition of claim1 wherein said microporous structure is cellular and is characterized bya C/P ratio of from about 2 to about 200, an S value of from about 1 toabout 30, and an average cell size from about 0.5 to about 100 microns.4. The composition of claim 1 wherein said microporous polymer structureis isotropic and is characterized by an average pore diameter of fromabout 0.1 to about 5 microns and an S value of from about 1 to about 10.5. The composition of claim 1 wherein said organic solvent comprises analcohol having from 1 to 4 carbon atoms per molecule.
 6. A method forthe immobilization of lipase on a microporous structure comprising asynthetic hydrophobic thermoplastic aliphatic olefin polymer selectedfrom the group consisting of aliphatic olefinic polymers, oxidationpolymers, ionic polymers and blends thereof, said immobilization beingeffected without pretreatment of said structure or pretreatment by firstwetting said polymer with a polar water miscible organic solvent inwhich such polymer is insoluble and which does not deactivate saidlipase, and then soaking said support in a dilute aqueous solution ofsaid lipase.
 7. The method of claim 6 wherein said organic solventcomprises an alcohol having from 1 to 4 carbon atoms per molecule, saidsolution is pH adjusted from about 4.0 to about 7.0 and theconcentration of said lipase in said solution is such as to provide atleast about 35 lipase units per ml, but less than 500 lipase units perml.
 8. A process for the hydrolysis of liquid fats comprising contactingsaid fats in the presence of water at hydrolyzing conditions with lipaseimmobilized by adsorption from aqueous solution on a microporousstructure comprising a synthetic hydrophobic thermoplastic aliphaticolefin polymer selected from the group consisting of aliphatic olefinicpolymers, oxidation polymers, ionic polymers and blends thereof, saidstructure not having been pretreated prior to said adsorption or beingpretreated only by wetting with a polar water miscible organic solventin which said polymer is insoluble and which does not deactivate saidlipase.
 9. The process of claim 8 wherein said contacting occurs in acolumn packed with discrete particles of said porous polymeric supporthaving said lipase immobilized thereon.
 10. The process of claim 9wherein said liquid fats and said water are passed cocurrently throughsaid column in a direction parallel to the longitudinal axis of saidcolumn.
 11. The process of claim 9 wherein said liquid fats and saidwater are passed countercurrently through said column in directionsparallel to the longitudinal axis of said column.
 12. The process ofclaim 10 wherein said liquid fats and water are passed into said columnat one end and reaction products comprising glycerol and fatty acids areremoved at the opposite end.
 13. The process of claim 11 wherein saidcolumn is vertical with respect to its longitudinal axis, said liquidfats and said water being passed into said column at opposite endsthereof, said fats being passed into said column at the lower end, areaction product comprising fatty acids being removed from said columnat the upper end of said column, and a reaction product comprisingglycerol being removed from said column at the lower end thereof. 14.The process of claim 9 wherein the activity of said lipase with respectto the hydrolysis of said fats is restored by first flushing thecontents of said column with a solvent suitable for the removal of spentlipase and residual fats from said porous polymeric support, thenflushing the contents of said column with water to remove said solvent,then passing a broth of fresh lipase through said column and finallyflushing the contents of said column with water to remove excess lipase.15. The process of claim 8 wherein said contacting occurs in asuspension of said immobilized lipase in a liquid reaction mixturecomprising fats and water maintained by the continuous addition theretoof a stream of liquid fats and a stream of water, and by the continuouswithdrawal therefrom of a portion of said mixture containing reactionproducts comprising fatty acids and a glycerol solution.
 16. The processof claim 8 wherein said hydrolyzing conditions comprise a residence timeof from about 2 to about 60 hours, and a temperature high enough toenable said fat to be in liquid phase and less than the deactivatingtemperature of the lipase.
 17. The process of claim 8 wherein saidmicroporous structure is cellular and comprises a plurality ofsubstantially spherical cells having an average diameter from about 0.5to about 100 microns, distributed substantially uniformly throughout thestructure, adjacent cells being interconnected by pores smaller indiameter than said microcells, the ratio of the average cell diameter tothe average pore diameter being from about 2:1 to about 200:1, saidpores and said cells being void.
 18. The process of claim 8 wherein saidmicroporous structure is cellular and is characterized by a C/P ratio offrom about 2 to about 200, an S value of from about 1 to about 30, andan average cell size from about 0.5 to about 100 microns.
 19. Theprocess of claim 8 wherein said microporous polymer structure isisotropic and is characterized by an average pore diameter of from about0.1 to about 5 microns and an S value of from about 1 to about
 10. 20.The process of claim 8 wherein a concentration of glycerol is maintainedin said water of not greater than about 40 wt. %.
 21. A process for thehydrolysis of liquid fats comprising contacting said fats in thepresence of water at hydrolyzing conditions with lipase immobilized byadsorption from aqueous solution on a microporous structure comprising asynthetic hydrophobic thermoplastic aliphatic olefin polymer selectedfrom the group consisting of aliphatic olefinic polymers, oxidationpolymers, ionic polymers and blends thereof, said structure not havingbeen pretreated or having been pretreated prior to said adsorption onlyby wetting with a polar water miscible organic solvent in which saidpolymer is insoluble and which does not deactivate said lipase, saidcontacting being effected by means of a column packed with a powder ofsaid structure on which said lipase is immobilized, the flow of saidliquid fats and said water in said column being cocurrent, said powderbeing from about 150 to about 450 micron particle size.
 22. The processof claim 21 wherein said microporous structure is cellular and comprisesa plurality of substantially spherical cells having an average diameterfrom about 0.5 to about 100 microns, distributed substantially uniformlythroughout the structure, adjacent cells being interconnected by poressmaller in diameter than said microcells, the ratio of the average porediameter being from about 2:1 to about 200:1, said pores and said cellsbeing void.
 23. The process of claim 21 wherein said microporousstructure is cellular and is characterized by a C/P ratio of from about2 to about 200, an S value of from about 1 to about 30, and an averagecell size from about 0.5 to about 100 microns.
 24. The process of claim21 wherein said microporous polymer structure is isotropic and ischaracterized by an average pore diameter of from about 0.1 to about 5microns and an S value of from about 1 to about
 10. 25. A process forthe hydrolysis of fats comprising maintaining a lower liquid phase ofsaid fats and an upper liquid phase comprising water, said phases beingseparated at their interface with a horizontally disposed diaphragmcomprising three layers, the bottom most of said layers being ahydrophobic filter cloth, the middle layer being fibers of a supportcomprising a synthetic hydrophobic microporous thermoplastic aliphaticolefin polymer, said fibers of said support in said middle layer havinglipase immobilized thereon, the top most layer of said diaphragm being aretaining means capable of maintaining the fibers of said middle layerin place, said fats flowing upward through said bottom layer and intocontact with said supported lipase of said middle layer where in thepresence of water from said upper phase, and at hydrolyzing conditions,the hydrolysis of said fats occurs, the fatty acids product of saidhydrolysis rising to the surface of said upper phase to form a separateuppermost phase and the glycerol product of said hydrolysis dissolvingin said upper phase, said fatty acids being removed as said uppermostphase and glycerol products in aqueous solution being recovered fromsaid upper phase and additional fats and water being added as requiredto maintain the desired inventory of each.
 26. The process of claim 25wherein the activity of said lipase with respect to the hydrolysis ofsaid fats is restored by sequentially flushing said diaphragm with threeflushing liquids which enter said diaphragm at the top layer and exitthrough means provided to bypass the bottom layer, the first saidflushing liquid comprising a solution of water and a solvent suitablefor the removal of spent lipase from said support, the second flushingliquid comprising a broth of fresh lipase and the last liquid comprisingwater.
 27. The process of claim 25 wherein said hydrolyzing conditionscomprise a temperature high enough to enable said fat to be in liquidphase and less than the deactivating temperature of the lipase and aresidence time of about 20 minutes to about 60 minutes.
 28. The processof claim 25 wherein the average diameter of said fibers is about 3 toabout 7 microns.
 29. The process of claim 25 wherein the openings insaid hydrophobic filter cloth are from about 3 to about 5 microns insize.
 30. The process of claim 25 wherein said lipase being immobilizedon said support by adsorption from aqueous solution either withoutpretreatment or following pretreatment of said structure only by wettingwith a polar water miscible organic solvent in which said polymer isinsoluble and which does not deactivate said lipase.
 31. The process ofclaim 30 wherein said microporous structure is cellular and comprises aplurality of substantially spherical cells having an average diameterfrom about 0.5 to about 100 microns, distributed substantially uniformlythroughout the structure, adjacent cells being interconnected by poressmaller in diameter than said microcells, the ratio of the average celldiameter to the average pore diameter being from about 2:1 to about200:1, said pores and said cells being void.
 32. The process of claim 30wherein said microporous structure is cellular and characterized by aC/P ratio of from about 2 to about 200, an S value of from about 1 toabout 30, and an average cell size from about 0.5 to about 100 microns.33. The process of claim 30 wherein said microporous polymer structureis isotropic and is characterized by an average pore diameter of fromabout 0.1 to about 5 microns and an S value of from about 1 to about 10.34. A process for the hydrolysis of fats comprising a series of at leasttwo stages in series, each stage comprising the phases and diaphragm asdefined in claim 25, the liquid flow in said series being considereddownstream with respect to the direction of flow of the non-aqueousphase from one stage to another in said series, the direction of flow ofthe aqueous phase from one stage to another being considered upstreamand countercurrent to said non-aqueous phase, the liquid fat to saidprocess being introduced into the lower phase of the first upstreamstage in said series and the water to said process being introduced intothe upper phase of the last downstream stage in said series, an aqueousstream from the upper phase of each stage being passed to the upperphase of the next upstream stage, except that the aqueous stream fromthe upper phase of said first upstream stage is withdrawn as theglycerol product stream, the non-aqueous stream from the uppermost phaseof each stage being passed to the lower phase of the next downstreamstage, except that the non-aqueous stream from the uppermost phase ofsaid last downstream stage is withdrawn as the fatty acid productstream.
 35. A process for the hydrolysis of fats comprising maintaininga suspension comprising lipase immobilized by adsorption from aqueoussolution on particles of a microporous structure comprising a synthetichydrophobic thermoplastic aliphatic olefin polymer and blends thereof,said structure having not being pretreated, or having been pretreatedprior to said adsorption only by wetting with a polar water miscibleorganic solvent in which such polymer is insoluble and which does notdeactivate said lipase, said suspension being in a liquid reactionmixture comprising fats and water by the continuous addition thereto ofa stream of liquid fats and a stream of water, and by the continuouswithdrawal therefrom of a portion of said mixture containing reactionproducts comprising fatty acids and a glycerol solution, theconcentration of lipase in said liquid reaction mixture being at leastabout 556 grams per liter of reactor volume per liter per hour of liquidfats added.
 36. The process of claim 35 wherein said hydrolyzingconditions comprise a residence time of from about 2 to about 60 hoursand a temperature sufficient to maintain liquid phase and less than thedeactivating temperature of the lipase.
 37. The process of claim 35wherein a stirring means is operated in said reaction mixture tomaintain said suspension.
 38. The process of claim 35 wherein saidportion of said reaction mixture withdrawn is passed through a filtermeans to enable recovery of said products and the retention of saidparticles in said reaction mixture.